Nuclear localization of the phosphatidylserine receptor protein via multiple nuclear localization signals

The interaction between phosphatidylserine and its receptor on phagocytic cells plays a critical role in the clearance of apoptotic bodies under normal physiological condition. A specific receptor for phosphatidylserine (PSR) has recently been identified by phage display and shown to mediate phosphatidylserine dependent phagocytosis. Here we show that the protein encoded by the PSR cDNA is localized in the nuclei through multiple nuclear localization signals. First, a fusion between PSR and GFP is localized in the nuclei of transfected cells, suggesting that PSR have intrinsic nuclear localization capability. Indeed, affinity-purified anti-PSR antibodies identified a 47 kDa protein species in cells transfected with untagged PSR and localized this protein in the nuclei by immunofluorescent confocal microscopy. In NIH3T3 cells, which express endogenous PSR mRNA, a similar 47 kDa species was detected and localized in the nuclei. Finally, multiple nuclear localization signals were identified in PSR sequence, each capable of targeting GFP to the nuclei. Together, these results suggest that PSR may serve a dual role both on the cell surface and in the nuclei.     

Overexpression of high molecular weight FGF-2 forms inhibits glioma growth by acting on cell-cycle progression and protein translation

In order to clarify the role of HMW FGF-2 in glioma development and angiogenesis, we over-expressed different human FGF-2 isoforms in C6 rat glioma cell line using a tetracycline-regulated expression system. Phenotypic modifications were analyzed in vitro and compared to untransfected cells or to cells over-expressing 18 kDa FGF-2 or all FGF-2 isoforms. In particular, we demonstrate that HMW FGF-2 has unique features in inhibiting glioma cell proliferation. HMW FGF-2 expressing cells showed a cell-cycle arrest at the G2M, demonstrating a role of HMW FGF-2 in controlling the entry in mitosis. Moreover, hydroxyurea was ineffective in blocking cells at the G1S boundary when HMW FGF-2 was expressed. We also show that the HMW FGF-2 isoforms inhibit 4E-BP1 phosphorylation at critical sites restoring the translation inhibitory activity of 4E-BP1. In vivo, inhibition of tumor growth was observed when cells expressed HMW FGF-2. This indicates that HMW FGF-2 inhibits tumor growth in glioma cells by acting on cell-cycle progression and protein translation.     

Nuclear localization signal of ING4 plays a key role in its binding to p53

ING4, a novel member of ING family, is recently reported to interact with tumor suppressor p53 and negatively regulate the cell growth with significant G2/M arrest of cell cycle in HepG2 cells through upregulation of p53-inducible gene p21. However, which region of ING4 could have contributed to the binding to p53 remains largely unclear. Herein, the GST-pulldown experiments revealed that the middle region of ING4, a potential bipartite nuclear localization signal (NLS), could be involved in the binding to p53. Furthermore, the interaction of ING4 to p53 was abrogated in vitro and in vivo when certain mutations or the entire deletion of the NLS domain occurred. More interestingly, the mutations of the NLS domain could alter the ING4 nuclear localization, disrupt the interaction of ING4 with p53, and even, deregulate the p53-inducible gene p21 in MCF-7 cells. All data indicated that the NLS domain of ING4 is essential for the binding of ING4 to p53 and the function of ING4 associated with p53.     

Detection of two high molecular weight hydrophobic forms of the human estrogen receptor

The human estrogen receptor gene encodes a single protein of molecular weight 65,000 daltons. However, using a sensitive and rapid technique of high-performance hydrophobic interaction chromatography we have detected two distinct estrogen receptor species both of which are high molecular weight proteins (ca. 60A) as determined by high-performance size-exclusion chromatography. These are detected either in the presence or absence of sodium molybdate; rechromatography of individual isoform indicates that the two protein complexes have independent hydrophobic contact points. Consistent elution patterns of the two receptor species indicates they are formed selectively. We conclude that different post-translational modifications of the estrogen receptor protein could allow their specific interaction with non-receptor components resulting in the formation of two distinct high molecular weight complexes which would be rapidly resolved by high-performance hydrophobic interaction chromatography.     

Nuclear localization of the DOCK180/ELMO complex

DOCK180 protein plays a key role during development, cell motility, and phagocytosis. It forms a complex with another protein ELMO, and this complex acts as a guanine nucleotide exchange factor (GEF) for Rac. However, DOCK180-containing complexes have not been purified by unbiased biochemical approaches, and the nature and subcellular localization of these complexes remain unclear. Here, we show that a large fraction of endogenous DOCK180 is present as a 700kDa nuclear complex with ELMO proteins. In addition, this nuclear DOCK180/ELMO complex has functional Rac-GEF activity. Furthermore, endogenous DOCK180 could be found in complexes with different ELMO isoforms (ELMO1, 2 or 3) in different cell lines, depending on the ELMO isoforms expressed. These studies suggest that DOCK180 may associate with different ELMO proteins to form cell-type specific complexes and may have functions in both the nucleus and the cytoplasm.     

Structural requirements in heparin for binding and activation of FGF-1 and FGF-4 are different from that for FGF-2

Size- and structure-defined oligosaccharides from heparin, 2-O-desulphated(2-O-DS-) heparin, 6-O-desulphated (6-O-DS-) heparin, carboxy-reduced(CR-) heparin, and carboxyamidomethylsulphonated (AMS-) heparinwere utilized in characterizing the structural properties ofheparin to specifically bind to basic fibroblast growth factor(FGF-2) and to modulate the mitogenic activity of FGF-2 (Ishihara,M.et al., Glycobiology, 4, 451–458, 1994). The previousresults showed that both 2-O-sulphate groups and the negativecharge of the carboxy group in iduronate residues are requiredfor specific interaction with FGF-2, but the 6-O-sulphate groupsin N-sulphated glucosamine (GlcNS) residues do not influencethe interaction with FGF-2. In the present study, the same oligosaccharideswere fractionated on a FGF-1- or FGF-4-affinity column, andwere assessed as promotors of FGF-1- or FGF-4-induced proliferationof adrenocortical endothelial (ACE) cells and chlorate-treatedACE cells. The present results suggest that the smallest heparin-derivedoligosaccharide binding to these growth factors with the highestaffinity and promoting their mitogenic activities is a fullyN-sulphated decasaccharide enriched in 2-O- and 6-O- sulphateddisaccharide units. In contrast to our results with FGF-2, ahigh content of 6-O-sulphate groups in GlcNS residues is requiredfor specific interaction with FGF-1 and FGF-4. FGF-1 FGF-4 heparin heparan sulphate oligosaccharides     

A high molecular weight actin binding protein: Its localization in the cortex of the sea urchin egg

From the Triton-treated cortex fraction of sea urchin eggs, a high molecular weight actin binding protein (260K protein) was solubilized by a high salt solution and purified. A cosedimentation assay revealed that the 260K protein binds to actin filaments in a concentration-dependent manner. The low-shear viscosity of actin solutions largely increased in a concentration-dependent manner after addition of 260K protein. Electron microscopy showed that this protein induces the formation of large curled bundles of actin filaments. Different from fascin-induced actin bundles, no clear striations were observed within the actin bundles formed by the 260K protein. Antibodies against the 260K protein were raised in a rabbit and affinity purified. Immunoblotting analysis of Triton-solubilized cortex and various subcellular fractions showed that first only a single band reacted with the antibody and second that the 260K protein exclusively localized in the cortex fraction. Indirect immunofluorescence microscopy localized the protein in the cortex and the region of the cleavage furrow. After double staining, the fluorescence images for actin filaments and the 260K protein well correlate with each other.     

Impact of IgG2 high molecular weight species on neonatal Fc receptor binding assays

A cell-based assay and a solution neonatal Fc receptor (FcRn) binding assay were implemented for the characterization of an IgG₂ antibody after observation that different product lots exhibited unexpected differences in FcRn binding in the cell-based format with membrane-bound FcRn. The experiments described here suggest that the apparent differences observed in the FcRn binding across different product lots in the cell-based format can be attributed to the different levels of the higher order high molecular weight species (HMWs) in them. A strong correlation between FcRn binding in the cell-based format and the percentage (%) higher order HMWs suggests that small amounts (∼0.1%) of the latter could cause the enhanced apparent FcRn binding (% relative binding ranging from 50 to 100%) in the format. However, when the binding was assessed with recombinant FcRn in soluble form, avidity effects were minimal and the assay format exhibited less sensitivity toward the differences in higher order HMWs levels across product lots. In conclusion, a solution-based assay may be a more appropriate assay to assess FcRn binding of the dominant species of an Fc-fusion protein or monoclonal antibody if minor differences in product variants such as higher order HMWs are shown to affect the binding significantly.     

Chromatin compaction and cell death by high molecular weight FGF-2 depend on its nuclear localization, intracrine ERK activation, and engagement of mitochondria

Fibroblast growth factor 2 (FGF-2) is produced as CUG-initiated, 22-34 kDa or AUG-initiated 18 kDa isoforms (hi- and lo-FGF-2, respectively), with potentially distinct functions. We report that expression of hi-FGF-2 in HEK293 cells elicited chromatin compaction preceding cell death with apoptotic features. Nuclear localization of the intact protein was required as expression of a non-nuclear hi-FGF-2 mutant failed to elicit chromatin compaction. Equally ineffective, despite nuclear localization, was the over-expression of the 18 kDa core sequence (lo-FGF-2). Chromatin compaction by hi-FGF-2 was accompanied by increased cytosolic cytochrome C, and was attenuated either by over-expression of Bcl-2 or by a peptide inhibitor of the pro-apoptotic protein Bax. In addition hi-FGF-2 elicited sustained activation of total and nuclear extracellular signal regulated kinase (ERK1/2) by an intracrine route, as it was not prevented by neutralizing anti-FGF-2 antibodies. Inhibition of the ERK1/2 activating pathway by dominant negative upstream activating kinase, or by PD 98059, prevented chromatin compaction by hi-FGF-2. ERK1/2 activation was not affected by the Bax-inhibiting peptide suggesting that it occurred upstream of mitochondrial involvement. We conclude that the hi-FGF-2-induced chromatin compaction and cell death requires its nuclear localization, intracrine ERK1/2 activation and mitochondrial engagement.     

Mapping of caldesmon: relationship between the high and low molecular weight forms

Caldesmon is a widely distributed contractile protein that occurs in both a high molecular weight [120-150-kilodalton (kDa)] and a low molecular weight (71-80-kDa) form, depending on the tissue. The structural relationship between these two forms was examined by mapping techniques. Partial cyanogen bromide cleavage in conjunction with sodium dodecyl sulfate gel electrophoresis was used to construct a map of the cleavage points and determine the relative position of the fragments in a high molecular weight caldesmon from chicken gizzard (caldesmon125). By use of this map, markers for different regions of the protein were obtained: Antibodies directed toward certain areas were prepared by affinity purification, and specific 125I-labeled tryptic peptides were found to originate from terminal cyanogen bromide fragments. Mapping of a lower molecular weight form of caldesmon (caldesmon72 from chicken liver) revealed the presence of sequences located in both ends of caldesmon125. A terminal 38-kDa fragment of both proteins was apparently identical on the basis of arrangement of cleavage sites, antibody reactivity, and iodopeptide mapping. Fragments from the other end of both proteins exhibited an identical pattern of peptides. These results show that it is sequences located in the central area of caldesmon125 which are missing in caldesmon72, indicating that the smaller molecule is not simply a proteolytic product of the larger. The two forms of caldesmon may be derived from separate genes or by alternative splicing from a single gene.     

The N-terminus of porcine circovirus type 2 replication protein is required for nuclear localization and ori binding activities

Porcine circovirus type 2 possesses a circular, single-stranded DNA genome that requires the replication protein (Rep) for virus replication. To characterize the DNA binding potential and the significant region that confers the nuclear localization of the Rep protein, the defined coding regions of rep gene were cloned and expressed. All of the recombinant proteins except for the N-terminal 110 residues deletion mutant could bind to the double-stranded minimal binding site of replication origin (ori). In addition, the N-terminal deletion mutant lacking 110 residues exhibited mainly cytoplasmic staining in the transfected cells in contrast to the others, which localized dominantly in the nucleus, suggesting that this N-terminal domain is essential for nuclear localization. Furthermore, a series of green fluorescence proteins (GFP) containing potential nuclear localization signal (NLS) sequences were tested for their cellular distribution. The ability of the utmost 20 residues of the N-terminal region to target the GFP to the nucleus confirmed its role as a functional NLS.     

Further characterization of the interaction of histidine-rich glycoprotein with heparin: evidence for the binding of two molecules of histidine-rich glycoprotein by high molecular weight heparin and for the involvement of histidine residues in heparin binding

Rabbit histidine-rich glycoprotein (HRG, 94 kDa) binds heparin with high affinity (apparent Kd 60-110 nM). Eosin Y (1 equiv) bound to HRG was used as a reporter group to monitor associations of HRG with heparins of molecular mass 10, 17.5, and 30 kDa. The stoichiometries of the heparin-HRG complexes were determined by fluorescence and absorbance measurements as well as by analytical ultracentrifugation. Two types of complex form: complexes of 1 heparin:1 HRG and of 1 heparin:2 HRG. The 1:2 complex formation requires a minimum heparin chain length since 17.5-kDa but not 10-kDa heparin binds two HRG molecules. The formation of the 1:2 complexes of the larger heparin fractions is enhanced by divalent copper or zinc (1-10 equiv) bound to HRG. However, metal is not required for complex formation since all sizes of heparin examined interact tightly with HRG in the presence of ethylenediaminetetraacetic acid. Between 0.1 and 0.3 M ionic strength, both 1:1 and 1:2 complexes of heparin with HRG are progressively destabilized. No heparin-HRG complex is found at ionic strengths of 0.5 M. Between pH 8.5 and pH 6.5 both 1:2 and 1:1 complexes are found with 17.5-kDa heparin, but at pH 5.5 only 1:1 complexes are formed. The heparin-HRG interaction is progressively decreased by modification of the histidine residues of HRG, whereas modification of 22 of the 33 lysine residues of HRG has little effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nuclear and nucleolar localization signals and their targeting function in phosphatidylinositol 4-kinase PI4K230

PI4K230, an isoform of phosphatidylinositol 4-kinase, known primarily as a cytoplasmic membrane-bound enzyme, was detected recently also in the nucleolus of several cells. Here we provide mechanistic insight on the targeting function of its putative nuclear localization signal (NLS) sequences using molecular modeling, digitonin-permeabilized HeLa cells and binding to various importins. The synthetic sequence ⁹¹⁶NFNHIHKRIRRVADKYLSG⁹³⁴ comprising a putative monopartite NLS (NLS1), targeted covalently bound fluorescent BSA to the nucleoplasm via classical importin α/β mechanism employing importins α1 and α3 but not α5. This transport was inhibited by wheat germ agglutinin and GTPγS. The sequence ¹⁴¹⁴SKKTNRGSQLHKYYMKRRTL¹⁴³³, a putative bipartite NLS (NLS2) proved ineffective in nuclear targeting if conjugated to fluorescently labeled BSA. Nonetheless, NLS2 or either of its basic clusters directed to the nucleolus soybean trypsin inhibitor that can pass the nuclear pore complex passively; moreover, an expressed 58 kDa fragment of PI4K230 (AA1166–1667) comprising NLS2 was also imported into the nucleus by import factors of reticulocyte lysate or by importin α1/β or α3/β complexes and localized to the nucleolus. We conclude that the putative bipartite NLS itself is a nucleolar targeting signal, and for nuclear import PI4K230 requires a larger sequence around it or, alternatively, the monopartite NLS.     

Effect of low molecular weight heparin and different heparin molecular weight fractions on the activity of the matrix-degrading enzyme aggrecanase: structure-function relationship

The matrix-degrading enzyme aggrecanase has been identified in cartilage and is largely responsible for cartilage breakdown. The present study determined the efficacy of different heparin molecular weight fractions (HMWFs) and low molecular weight heparins (LMWHs) on aggrecanase activity. Aggrecanase activity was determined using biotinylated peptide substrate, which was immobilized onto streptavidin-coated 96-well plates; aggrecanase enzyme was then added. Proteolysis of the substrate at the specific amide bond was detected using specific antibody for the neoepitope generated. HMWFs ranging from 1,700 to 12,000 Da demonstrated a concentration-dependent inhibitory efficacy of aggrecanase activity, with a Ki ranging from 5,000 nM down to 1 nM as a function of the molecular weight. The higher the molecular weight distribution, the greater the inhibitory efficacy of the heparin fragments toward aggrecanase activity. The absence or presence of antithrombin did not alter the affinity of heparin in inhibiting aggrecanase. Additionally, tissue factor pathway inhibitor at various levels did not alter the activity of aggrecanase. LMWHs demonstrated different levels of potency in inhibiting aggrecanase activity as a function of their average molecular weight distribution. Tinzaparin (average molecular weight = 6,500 Da) and enoxaparin (average molecular weight = 4,500 Da) demonstrated a Ki of 20 and 80 nM, respectively. The aggrecanase inhibitory effect of LMWH might contribute to blocking inflammation and tumor invasion by inhibiting aggrecanase activity and maintaining an intact extracellular matrix barrier.